Project description:This study uses five species of the genus Ecrobia as a model taxon to demonstrate the applicability of proteomic fingerprinting measured by MALDI-TOF MS (matrix-assisted laser/desorption ionization time-of-flight mass spectrometry) to cryptic gastropod species and evaluate the discriminative power the proteomic profiles.
Project description:Ageing is associated with a range of chronic diseases and has diverse hallmarks. Mitochondrial dysfunction is implicated in ageing, and mouse-models with artificially enhanced mitochondrial DNA (mtDNA) mutation rates show accelerated ageing. A scarcely studied aspect of ageing, because it is invisible in aggregate analyses, is the accumulation of somatic mtDNA mutations which are unique to single cells (cryptic mutations). We find evidence of cryptic mtDNA mutations from diverse single-cell datasets, from three species, and discover: cryptic mutations constitute the vast majority of mtDNA mutations in aged post-mitotic tissues, that they can avoid selection, that their accumulation is consonant with theory we develop, hitting high levels coinciding with species specific mid-late life, and that their presence covaries with a majority of the hallmarks of ageing including protein misfolding and ER stress. We identify mechanistic links to ER stress experimentally and further give an indication that aged brain cells with high levels of cryptic mutations show markers of neurodegeneration and that calorie restriction slows the accumulation of cryptic mutations.
Project description:Biological Relevance and Intent: Cryptic species within the Aspergillus section Fumigati, such as the Aspergillus viridinutans species complex, are increasingly recognized as important opportunistic pathogens in both human and veterinary medicine. These species often exhibit an intrinsic reduction in susceptibility to standard azole antifungals. The intent of this experiment was to determine how the tolerant veterinary isolate Aspergillus felis CCF5624 responds to acute azole stress (tolerance is transient state, not genetically determined like mutations). This study aimed to map the active cellular survival pathways and identify regulatory networks that could be targeted to bypass this protective tolerance machinery. Experimental Workflow Overview: Conidia suspensions of Aspergillus felis CCF5624 were inoculated onto pure RPMI medium covered with sterile cellophane and cultivated for 40 hours at 37 °C to establish vegetative colonies. To capture the immediate transcriptomic shifts associated with drug tolerance, the grown biomass was transferred directly to new RPMI plates supplemented with either a control solvent (DMSO) [abbreviated as \"A\"] or one of three clinical azole compounds (itraconazole, voriconazole, or posaconazole) [abbreviated as B, C,D, respectively] at concentrations corresponding to EUCAST resistance breakpoints. Following an acute 4-hour exposure, fungal biomass was harvested and flash-frozen in liquid nitrogen. Total RNA was isolated using a combined TRI reagent and spin-column methodology. High-throughput RNA sequencing (RNA-seq) was performed by Novogene Limited UK. Poly-A enriched mRNA libraries were prepared and sequenced on the Illumina NovaSeq X Plus platform using a 150 bp paired-end (PE150) strategy, yielding a minimum sequencing depth of 3 Gb (approximately 10 million paired-end reads) per individual sample. Reference gemone Aspergillus pseudoviridinutans IFM_55266 https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/018/340/605/GCF_018340605.1_Asppvi_assembly01/GCF_018340605.1_Asppvi_assembly01_genomic.fna.gz https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/018/340/605/GCF_018340605.1_Asppvi_assembly01/GCF_018340605.1_Asppvi_assembly01_genomic.gtf.gz
Project description:The full genome sequencing of the filamentous fungi Aspergillus nidulans, Aspergillus niger and Aspergillus oryzae has opened the possibilities for studying the cellular physiology of these fungi on a systemic level. As a tool to explore this, we are presenting an Affymetrix GeneChip developed for transcriptome analysis of any of the three above-mentioned aspergilli. Transcriptome analysis of triplicate batch cultivations of all three aspergilli on glucose-and xylose media has been performed, and used to validate the performance of the micro array. By doing gene comparisons of all three species, and cross-analysing this with the expression data, 23 genes, including the xylose transcriptional activator XlnR, have been identified to be a conserved response across the Aspergillus sp. Promoter analysis of the upregulated genes in all three species suggest the XlnR-binding site to be 5’-GGNTAAA-3’. We are thus presenting a validated tool for transcription analysis of three Aspergillus species and a methodology for comparative transcriptomics. Keywords: Physiological response